Carbapenem-resistant Enterobacterales among hospitalized patients in Cape Town, South Africa: clinical and microbiological epidemiology

Abstract Background Carbapenem-resistant Enterobacterales (CRE) are a substantial problem in Cape Town. CRE epidemiology is largely unknown and mortality remains high. Objectives To describe and characterize the clinical and microbiological epidemiology of CRE within Cape Town hospitals to better inform therapy with regard to current and novel antibiotics, as well as improve antimicrobial stewardship (AMS), and infection prevention and control (IPC). Methods This prospective, multicentre study performed between 1 November 2020 and 30 November 2022, across three public and three private hospitals included hospitalized participants with CRE from clinical cultures. Participant demographics, clinical information and microbiology results were collected and analysed. Results Ninety percent of participants were from public hospitals. The age distribution ranged from 7 days to 88 years. Notable risk factors for CRE infection included recent exposure to antibiotics, medical devices and surgery. The most prevalent species was Klebsiella pneumoniae. However, a higher proportion of Serratia marcescens compared with previous reports was identified. The detected carbapenemases were blaOXA-48-like (80%) and blaNDM (11%). With the exception of amikacin (63%), tigecycline (65%), colistin (95%) and ceftazidime/avibactam (87%), susceptibility to antibiotics was low. Conclusions This study identified common risk factors for CRE infection and generated a description of carbapenemase enzymes, species distribution and antibiograms, enabling a better understanding of CRE epidemiology. This provides insights into transmission patterns and resistance determinants of CREs, beneficial to informing data-driven regional patient management, AMS and IPC strategies.


Introduction
In 2017, the WHO published a list of priority pathogens for which the development of effective antibiotics is urgently needed. 1 Carbapenem-resistant Enterobacterales (CRE) are listed as critical pathogens and until the new β-lactam/β-lactamase inhibitor combinations (BLICs) became available, colistin was the last line of defence against these pathogens. 1,2n South Africa, carbapenemase-producing Enterobacterales emerged in 2011 with the detection of NDM and Klebsiella pneumoniae carbapenemase (KPC).The detection of oxacillinase-48 (OXA-48) and one of its variants, OXA-181, followed in 2012. 3,4ational surveillance initially described a predominance of NDM (59%) followed by OXA-48 and its variants (29%), from 1503 CRE isolates between 2012 and 2015, mainly cultured from blood (25%) and urine (22%). 5Outbreaks of both large across-province and smaller institutional clonal outbreaks of OXA-181-producing K. pneumoniae, respectively, were also described between 2012 and 2016. 6,7Subsequent national surveillance of CRE bacteraemia between 2015 and 2018, from 895 isolates, demonstrated a shift in the predominant carbapenemase to OXA-48 and variants (52%), followed by NDM (34%). 8Recent national surveillance of CRE bacteraemia between 2019 and 2020 confirmed these findings in 1082 isolates, where the most common carbapenemase detected was still OXA-48 and variants (76.8%), although increasing in proportion.This was followed by NDM (21.1%). 9Whilst KPC 3 was one of the first carbapenemases to be described in South Africa, similar to IMI-2, 10 VIM, 6,11,12 GES 6,12,13 and IMP, 11,12 they have infrequently been identified locally.
K. pneumoniae has consistently been the most frequent species isolated from national surveillance.However, the proportion of K. pneumoniae isolated has increased from 60% to 80% between 2012 and 2020.Enterobacter cloacae complex (6%-14%) and Serratia marcescens (5%-6%) have also commonly been isolated. 5,8,9he number of CRE causing infection is rapidly increasing, the epidemiology continues to evolve, and the high mortality associated with these pathogens has escalated the situation to not only a public health priority, but also a clinical emergency. 2,8,9part from a few institutional reports, 7,14 the clinical, microbiological and molecular epidemiology of CRE in Cape Town is mostly unknown.Recently, marked increases in CRE have been noted at both public and private hospitals in the city, in line with the rest of South Africa.However, these increases in CRE have not been fully analysed, either with regard to transmission or genetic repertoire.
An in-depth understanding of the CRE epidemiology will allow for better-informed therapeutic interventions with regard to old and new antibiotics, as well as improved antimicrobial stewardship (AMS), by facilitating evidence-based empirical and directed therapy in our setting.Furthermore, effective infection prevention and control (IPC) interventions necessitate data-driven strategies.
Therefore, this study aims to address some of this need, by describing and characterizing the clinical, microbiological and molecular epidemiology of CRE within the Cape Town Metropole, across public and private hospitals.

Study design, setting and population
This was a prospective, multicentre study.Participants were recruited via convenience sampling between 1 November 2020 and 30 November 2022, from three participating public hospitals (Groote Schuur Hospital, Red Cross War Memorial Children's Hospital and Tygerberg Hospital) and three participating private hospitals (Mediclinic Panorama, Netcare Christiaan Barnard Memorial Hospital, Netcare Blaauwberg Hospital) in Cape Town, Western Cape, South Africa.Further information about each hospital is available in Table S1 (available as Supplementary data at JAC-AMR Online).

Inclusion criteria
Hospitalized participants from whom phenotypic CRE (resistant to ertapenem, imipenem or meropenem) was isolated from clinical cultures obtained from any anatomical site during the study period (Figure 1).

Exclusion criteria
Duplicate or new CREs isolated from an already recruited participant (irrespective of whether the duplicate or new CRE was isolated from the participant during a subsequent admission to a different participating hospital), and participants with CRE isolated from surveillance cultures (rectal swabs, stool) were excluded.Enterobacterales with intrinsic reduced susceptibility to the carbapenems, such as Proteus spp., Providencia spp.and Morganella spp. with isolated imipenem resistance were not considered as CRE organisms, and participants from whom these organisms were cultured were also excluded.

Bacterial identification and susceptibility testing methods
Bacterial isolates were cultured, identified and had routine susceptibility testing performed at microbiology laboratories serving the respective hospitals.Carbapenemase detection was also routinely performed at these laboratories with the RESIST-4 O.K.N.V. (Coris BioConcept, Belgium) lateral flow assay or the Xpert Carba-R (Cepheid, USA) PCR.These laboratories are accredited by the South African National Accreditation System and include two public laboratories [National Health Laboratory Service (NHLS) laboratories at Groote Schuur Hospital and Tygerberg Hospital] and two private laboratories (Pathcare and Ampath).The study group did not perform confirmatory testing of isolate identification, routine susceptibility results or carbapenemase results.
Identification of bacterial isolates was performed using the VITEK 2 automated system (bioMérieux, France) or the MALDI-TOF VITEK MS (bioMérieux) platform.Susceptibility testing was performed using the VITEK 2 automated system (bioMérieux), Kirby-Bauer disc diffusion, ETEST (bioMérieux) gradient diffusion, or broth microdilution methods where relevant.Results of antibiotic susceptibility tests were interpreted using the CLSI (NHLS and Pathcare laboratories) or the EUCAST (Ampath laboratories) guidelines for the relevant year.Ceftazidime/avibactam ETEST (bioMérieux) MIC and colistin broth microdilution MIC were performed on selected isolates by participating laboratories.When these specific MIC results were not available from participating laboratories, the study group referred the isolates to Ampath laboratories for testing.

Informed consent
In-person or telephonic informed consent from participants or legal guardians, in the case of minors, was obtained.Similarly, informed consent was obtained from relatives when participants were too unwell to give consent, or had died.

Data collection and statistical analysis
Participant demographics, clinical information (including outcome status within 90 days after discharge) and microbiology results of bacterial isolates were prospectively collected from both the participant and review of paper-based and electronic medical and laboratory reports.Data were collected on a participant questionnaire and folder review document and entered into an electronic database (REDCap) for analysis.Data were analysed using STATA version 14 (College Station, TX, USA).
A descriptive analysis was performed on the demographics and clinical epidemiology of participants and the microbiological characteristics of bacterial isolates.Categorical variables were summarized using proportions and percentages, and continuous variables were summarized as medians with IQRs.Univariable and multivariable logistic regression analysis was performed to investigate factors that may be associated with in-hospital mortality.A sample size estimate for the logistic regression analysis was not performed.Variables were selected a priori based on the likelihood to contribute to death.Variables were included in the multivariable analysis if the P value was ≤0.2 in the univariable analysis.A P value of 0.05 was regarded as statistically significant.

Ethics
This study adhered to the principles outlined in the Declaration of Helsinki.Ethical approval for the study (HREC 096/2020), as well as for an isolate and clinical data biorepository (HREC R009/2020), was obtained from the University of Cape Town Human Research Ethics Committee.Ethical approval was also obtained from Stellenbosch University Health Research Ethics Committee as part of a reciprocal review (N21/01/ 002_RECIP_UCT_096/2020).

Results
Clinical, epidemiological and microbiological data were collected from 117 patients during the study period (Table 1).Ninety percent (n = 105/117) of study participants were from three public hospitals, with the remainder from three private hospitals (10%, n = 12/117).

Demographics and clinical characteristics
A description of the study population is presented in Table 1.Fifty-one percent (n = 60/117) were male participants.The age distribution ranged from 7 days to 88 years.The overall median age was 41 years (IQR 10-53).Children younger than 10 years accounted for 25% of the study population and the median age of this population was 122 days (IQR 43-676).The median time from admission to positive CRE sample collection was 15 days (IQR 8-35).In participants where data were available, 48% (n = 52/109) of participants were in the ICU at the time of positive CRE sample collection.Fifty-one percent (n = 55/108) had at least one underlying medical condition, of which the most common were HIV infection (31%, n = 17/55), TB (29%, n = 16/55) and chronic kidney disease (25%, n = 14/55).Forty-four percent of participants (n = 45/103) had previously been admitted to hospital, with 45% (n = 20/45) having at least two previous hospital admissions.Eighty-five percent (n = 88/105) of participants had at least one surgical procedure within 1 year prior to the positive CRE sample collection, with gastrointestinal tract surgery (51%, n = 45/88) being the most common.Almost all participants had received antibiotics within 30 days prior to their current admission or positive CRE sample collection (99%, n = 108/109).Most participants (89%, n = 95/107) had at least one medical device in situ at any time from admission to the time of positive CRE sample collection.Urine catheters (80%, n = 76/95) and central venous lines (68%, n = 65/95) were the most common.An MDRO had been previously isolated from thirty-three percent  Tootla et al.
(n = 39/117) of participants, either from a clinical sample or from a surveillance sample, within the year prior to the current positive CRE sample.Gram-negative MDROs were the most common previously isolated (95%, n = 37/39) organisms, of which the most frequent was CRE (78%, n = 28/39).No VRE were previously isolated, but MRSA had been isolated from two patients (5%, n = 2/39) within the year prior to the current positive CRE sample.Most participants lived in formal housing with a plumbed water supply and indoor toilet (86%, n = 98/114) and had no interprovince (interstate) or international travel in the year prior to the positive CRE sample collection (96%, n = 106/110).

Mortality and outcome
The all-cause in-hospital mortality rate was 29% (n = 33/115).The median time from positive CRE sample collection to death was 27 days (IQR 14-38).
In the univariable logistic regression analysis, being in the ICU at the time of CRE sample collection, previous isolation of an MDRO and isolation of CRE from a sterile sample significantly increased the odds of in-hospital mortality.However, this significance was not confirmed by multivariable logistic regression analysis (Table 3).

Discussion
Recent publications on the epidemiology of CRE bacteraemia from three public teaching hospitals in the Western Cape

Epidemiology of CRE in Cape Town
demonstrated an increase in the number and proportion of cases between 2015 and 2018 (7% n = 112/1601) and between 2019 and 2020 (14% n = 298/2144). 8,9][17] Similar to previous studies in South Africa, 5,8,9 and globally, 15-17 the most prevalent CRE organism in this study was K. pneumoniae.However, of concern is the higher proportion of S. marcescens identified in our study (11%), compared with what has previously been described in South Africa (5%-6%), 4,16 as this organism is intrinsically resistant to colistin, further limiting therapeutic options.The only detected carbapenemases were bla OXA-48-like and bla NDM , of which bla OXA-48-like was the most prevalent.This is consistent with previous epidemiology suggesting that detection of bla VIM , bla IMP and bla KPC is uncommon in our setting. 5,8,9Differentiation of these enzymes is important to guide the use of ceftazidime/ avibactam, which has recently been registered in South Africa.To preserve its longevity, updated country-specific recommendations are to steward its use within a framework that recognizes the current predominant resistance mechanisms in carbapenemresistant Gram-negative bacteria. 2ith the exception of amikacin, tigecycline and colistin, overall susceptibility to all other non-β-lactam antibiotics that were tested was low, in keeping with what has recently been described. 9In contrast, the proportion of isolates in this study with imipenem and meropenem MICs of >8 mg/L was higher than recently described (30% and 42% of isolates, respectively, compared with 23% and 36% of isolates, respectively) in national surveillance of CRE isolates causing bacteraemia from 2019 to 2020. 9This increase in the proportion of isolates with MICs of >8 mg/L is of serious clinical concern as this excludes imipenem and meropenem as potential treatment for CRE infections, where country-specific stewardship recommendations include them in select patients when the MICs are ≤ 8 mg/L. 2 Although colistin MICs were ≤2 mg/L in almost all isolates, similar to amikacin and tigecycline, colistin usage for CRE infection is limited due its side-effect profile, dosing and administration challenges, and questionable therapeutic efficacy. 2,18Notably, 13% of isolates (excluding NDM-producing isolates, n = 13/99) were resistant to ceftazidime/avibactam.In these isolates no carbapenemases were detected (n = 4) or tested for (n = 9).All tested isolates with only OXA-48-like carbapenemase detected (n = 55) were susceptible to ceftazidime/avibactam.Given that ceftazidime/avibactam has recently been registered in South Africa, and is only effective against specific resistance mechanisms, it is crucial that any gaps in knowledge around resistance-conferring mechanisms to novel antibiotics are identified with ongoing surveillance of susceptibility.Similarly, once the new siderophore cephalosporin cefiderocol becomes registered and available for use in South Africa, resistance and resistance-conferring mechanisms to this antibiotic will have to be monitored.
The most notable risk factors for CRE infection in this study included recent exposure to antibiotics, medical devices or surgery.Although AMS programmes have proved valuable in decreasing antibiotic consumption, antibiotic use in low-and middle-income countries is still comparatively higher than in high-income countries and improved context-based strategies to implement and optimize AMS are needed. 19Additionally, optimal management of medical devices (including reducing inappropriate use) should be effectively incorporated into AMS programmes.
Notably, 25% of the participants with CRE infections were children less than 10 years old.Whilst CRE infections are difficult to treat in adults, this is further exacerbated by limited safety, efficacy and dosing data in children for the 'last-line antibiotics' such as tigecycline and colistin and newer BLICs such as ceftazidime/ avibactam. 18Additionally, the use of IPC measures such as contact precautions and isolation limits physical movement and social Tootla et al.
interactions with staff, carers, siblings and other children, and adversely impacts children. 20This highlights that current 'adultcentric' treatment options and IPC strategies are even more challenging with paediatric populations, especially in resourceconstrained environments such as ours.

Limitations
Whilst this study provided valuable insights into the epidemiology of CRE in Cape Town, there were several limitations and challenges.The impact of the SARS-CoV-2 pandemic on voluntary hospital participation and required approval processes, as well as participant recruitment, was substantial.This resulted in very few private hospitals volunteering to participate in the study.Additionally, participant recruitment was severely impacted due to additional IPC measures implemented in response to the pandemic, which included restriction of study-related research and movement of study-related-staff within participating institutions.Hence, whilst recruitment of participants started in 2020, only a limited number of participants were recruited in 2020 (3%, n = 4/117), with more participants recruited in 2021 (22%, n = 26/117) and most participants recruited in 2022 (74%, n = 87/117).Most of the participants were recruited from the large academic teaching public hospitals.The public hospitals included in the study comprised all three main academic teaching hospitals in Cape Town and the data are most representative of this sector.

Epidemiology of CRE in Cape Town
Therefore, the true burden of CRE in the private sector and within smaller non-academic public hospitals is under-represented in this study.Similarly, the impact of the additional IPC measures may have also influenced CRE transmission dynamics within hospitals, as well as patient travel within and outside of South Africa.
A sample size estimate for variables associated with all-cause mortality in the logistic regression analysis was not performed and may have led to under-or over-representation of this data.
Laboratory-related limitations include not confirming all isolate identification, susceptibility and carbapenemase results with a standardized methodology.Likewise, the usage of both CLSI and EUCAST for susceptibility interpretation, depending on which microbiology laboratory was used, reflects real-life practice in Cape Town.However, it is unlikely that this would have affected the reported antibiogram as most isolates were from laboratories where CLSI criteria are used routinely.Furthermore, routine carbapenemase testing resulted in only 68% of isolates being tested, again reflecting the impact of non-standardized and variable reallife laboratory processing and practices.The CRE incidence or prevalence rate was not calculated, which might have served as a better indicator to evaluate the impact of AMS and IPC interventions.Lastly, despite being a prospective study, due to our medical records and prescription charts predominantly being paperbased, missing data were inevitable and could have affected the demographic and clinical epidemiology, highlighting the need for fully integrated electronic medical record keeping systems.

Conclusions
Despite the limitations, the study generated a description of carbapenemase enzymes and species distribution, enabling a better understanding of the epidemiology and risk factors for CRE.The information should inform data-driven regional patient management, AMS and IPC strategies, which could be beneficial to both public and private healthcare systems.Curtailing the spread and acquisition of CRE necessitates a comprehensive, coordinated and integrated approach including further studies of the determinants and rates of gastrointestinal colonization with CRE.

Table 1 .
Clinical and epidemiological characteristics of study participants

Table 3 .
Univariable and multivariable analysis of variables associated with in-hospital mortality in hospitalized patients with CRE a Urine, tracheal aspirate, sputum, pus/pus swab, central venous catheter tip.b Blood culture, tissue, fluid.